Functional nucleic acids, such as siRNAs, nucleic acid aptamers, and decoy nucleic acids, have drawn attention as pharmaceuticals or diagnostic agents in recent years, and research on and development of a variety of nucleic acid pharmaceuticals and the like are in progress with the goal of establishing medical applications for the same all over the world.
However, nucleic acids are generally problematic in that they are likely to be degraded by nucleolytic enzymes, such as nucleases, in vivo. In particular, siRNAs or RNA aptamers that have recently drawn attention as nucleic acid pharmaceuticals because of the applicability and effects thereof are composed of RNAs, which are very unstable in vivo. Accordingly, in vivo stability of nucleic acid is essential for the efficient and continuous exertion of the pharmacological effects of nucleic acid pharmaceuticals.
Many methods aimed at nucleic acid stabilization and stabilized nucleic acids have heretofore been reported. An example is a method for stabilization of decoy nucleic acids using a dumbbell shape (WO 2003/091432, WO 2005/014810, and US 2003/040613). Formation of a dumbbell-shaped nucleic acid is a method in which both ends of a double-stranded nucleic acid fragment are ligated to each other with a loop structure, such as a linker nucleic acid, to form a closed circle, so that the double-stranded nucleic acid fragment acquires resistance to degradation by a nucleolytic enzyme. This method, however, disadvantageously necessitates a process of cyclization of a linear nucleic acid fragment.
Use of artificially constructed nucleic acid analogues that are not be degraded by nucleases is also taken into consideration. However, the application of nucleic acid analogues that are not degraded in vivo for pharmaceutical products remains problematic from the viewpoint of safety, such as with regard to side effects.
Accordingly, development of nucleic acid pharmaceuticals that are composed to as a great extent as possible of naturally occurring nucleic acids, resistant to degradation by nucleolytic enzymes, stably maintained in vivo, and easily prepared in a simple and cost-effective manner has been awaited. To this end, a method that allows easy stabilization of a variety of nucleic acid fragments in a cost-effective manner is necessary.